The long-term goals of this project are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of seveal enzymes and green fluorescent proteins (GFPs). Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids, and membranes. Variations in the magnitude and direction of electric fields can significantly affect the rates of elementary processes such as electron and proton transfer, where charge moves over a substantial distance. Similarly, the transition states for many enzyme-catalyzed reactions involve a change in the distribution of charge relative to the starting material and/or products, and the selective stabilization of charge-separated transition states is essential for catalysis. The contours of electric fields steer the binding of substrates, inhibitors and allosteric effectors to macromolecules and directly affect binding constants. On a larger scale, electrostatic interactions affect protein folding, macromolecular interactions, and the assembly of subunits into larger structures. The magnitudes of the electric fields in proteins and the variations in thee fields at different sites are predicted to be enormous, but it is a challenge to obtain quantitativ experimental information on either local variations in electric fields in proteins or the time-dependent changes in these fields coupled to functionally relevant changes in charge distribution. The proposed research outlines a series of approaches and targets that can address these core issues. Aim 1 outlines development of methodology for introducing and characterizing vibrational probes for electric fields in proteins. These methods are used to probe fields in several enzymes. The proposed work focuses on a rigorous comparison between measured and calculated fields and incisive studies of the role of electrostatic interactions in catalytic mechanisms. Aim 2 outlines strategies to understand the mechanism(s) of the recently discovered coupling between light-driven structural dynamics of peptide-protein re-assembly or dissociation in split GFP. A wide range of structural and spectroscopic techniques will be deployed to elucidate the mechanism(s) of this coupling. The split semi-synthetic GFP system will also be used to probe the assembly of the -barrel itself using the built-in reporter chromophore, the origin(s) of color tuning, and both ground and excited state proton transfer. These are achieved by introducing unnatural amino acids as probes or perturbations at functionally interesting sites throughout the protein, now enabled by these semi-synthetic systems. Applications of these novel systems for advanced imaging and as modulators of enzyme function that affect cell physiology with light are described.